A Distinct Anti-EBV DNase Profile in Patients with Undifferentiated Nasopharyngeal Carcinoma Compared to Classical Antigens.

Nasopharyngeal cancer (NPC) is a prevalent type of cancer that often takes the form of undifferentiated carcinoma in the Maghreb region. It affects people of all ages. NPC diagnosis, mainly based on detecting Epstein-Barr virus (EBV), has not been well evaluated in North Africa. We compared the classical EBV serological tests using indirect immunofluorescence to the detection of EBV DNase antibodies by immunoblot in Algerian NPC patients. Significant variations were observed among different age groups of patients regarding the presence of VCA-IgA antibodies (0-14 and ≥30 years old, p < 0.0001; 15-19 and ≥30 years old, p < 0.01) and EA-IgA (0-14 and ≥30 years old, p < 0.01; 15-29 and ≥30 years old, p < 0.05). Differences were also noted in the titers of IgA anti-VCA and anti-EA antibodies across the three age groups. Some patients under the age of 30 with detectable IgG anti-VCA antibodies had undetectable IgA anti-VCA antibodies. These patients had a strong anti-DNase IgA response. However, older individuals had a higher level of anti-DNase IgG. Before treatment, children had strong DNase reactivity as indicated by specific IgA antibodies. Young adults had high IgA anti-DNase response, but the elderly (90.9%) had a lower response for these antibodies. Following therapy, the children retained high levels of IgA anti-DNase antibodies, and 66% of the young adults demonstrated robust antibody reactivity against DNase. In contrast, IgG responses to anti-DNase were low in children. This study demonstrated the utility of anti-DNase responses in the diagnosis and prognosis of NPC.

Plasma secretome analyses identify IL-8 and nitrites as predictors of poor prognosis in nasopharyngeal carcinoma patients.

Predicting tumor recurrence and death in patients with nasopharyngeal carcinoma (NPC) remains to date challenging. We here analyzed the plasmatic secretomes of NPC untreated and relapsing patients, and explored possible correlations with the clinical and pathological features and survival characteristics of the corresponding patient cohorts, with the aim of identifying novel prognostic biomarkers. This study included 27 controls, 45 untreated NPC and 11 relapsed patients. A set of 14 plasma cytokines were analyzed using Millipore multiplex assay. Nitrites were assessed by Griess method. A comparative analysis of each groups' secretome showed upregulation of IL-8, IL-12p70, IL-10 and IP-10 in untreated patients, and of IL-6, IL-10, MCP-1 and IP-10 in relapsing patients. Nitrites significantly correlated with IL-8 during relapse. Secretomes' network analyses revealed prevalence of high correlations between IL8/IL-17A and IFN-γ/IL12p70 in the control group, between TNF-α/IL-8/IL-6, TNF-α/VEGF/IFN-γ and IL-10/MCP-1 in the untreated group, and between IL-8/IL-6/IL-10, TNF-α/IL-8/IL-6, IL12-p70/VEGF/IL-10/IFN-γ, IL-6/IL-10/IFN-γ and IL-8/IP-10 in the relapse group. IL-12p70, IP-10 and MCP-1 levels respectively associated with gender, age and node metastasis respectively. Recurrence-free survival (RFS) analysis showed that patients presenting High IL-8/Low NO immunological scores presented a combined 80% probability of relapse/death after 53 months (combined log-rank test p = 0.0034; individual p = 0.012 and p = 0.016). Multivariate Cox hazard regression analysis revealed that IL-8 (HR = 7.451; 95% CI [2.398-23.152]; p = 0.001) and treatment type (HR = 0.232; 95% CI 0.072-0.749; p = 0.015) were independent prognostic factors. C&RT decision tree analysis showed that High IL-8/Low NO immunological scores predicted treatment failure in 50% cases starting the 36th month of follow-up (AUC = 1) for all of the studied cases and in 57% cases for patients receiving chemotherapy alone (AUC = 1). Altogether, our results showed that NPC development is accompanied with cytokines deregulation to form specific interaction networks at time of diagnosis and relapse, and demonstrate that High IL-8/Low NO signature may constitute a predictor of poor prognosis which may be useful to improve risk stratification and therapy failure management.

Silibinin down-regulates PD-L1 expression in nasopharyngeal carcinoma by interfering with tumor cell glycolytic metabolism.

The upregulation of checkpoint inhibitor PD-L1 expression has recently been associated with nasopharyngeal carcinoma (NPC) resistance to therapy. The mechanism of induction of PD-L1 has also been linked to enhanced aerobic glycolysis promoted by HIF1-α dysregulation and LDH-A activity in cancer. Here, we investigated the effect of the anti-tumoral compound Silibinin on HIF-1α/LDH-A mediated cancer cell metabolism and PD-L1 expression in NPC. Our results demonstrate that exposure to Silibinin potently inhibits tumor growth and promotes a shift from aerobic glycolysis toward oxidative phosphorylation. The EBV + NPC cell line C666-1 and glycolytic human tumor explants treated with Silibinin displayed a reduction in LDH-A activity which consistently associated with a reduction in lactate levels. This effect was accompanied by an increase in intracellular citrate levels in C666-1 cells. Accordingly, expression of HIF-1α, a critical regulator of glycolysis, was down-regulated after treatment. This event associated with a down-regulation in PD-L1. Altogether, our results provide evidence that silibinin can alter PD-L1 expression by interfering with HIF-1α/LDH-A mediated cell metabolism in NPC. These results provide a new perspective for Silibinin use to overcome PD-L1 mediated NPC resistance to therapy.

Expression Analysis of the Mediators of Epithelial to Mesenchymal Transition and Early Risk Assessment of Therapeutic Failure in Laryngeal Carcinoma.

Laryngeal squamous cell carcinoma (LSCC) is an aggressive malignancy which lacks early predictors of prognosis. Here, we hypothesized that expression and prognostic characterization of the critical mediators of epithelial to mesenchymal transition (EMT) may provide key information in this regard. Linear regression and multiple correspondence analyses were performed on immunohistochemical data obtained from 20 invasive tumors. Principal component and unsupervised hierarchical clustering were used to analyze the dataset patterns associating with LSCC metastatic profile. Survival and death risk assessments were performed using Kaplan-Meier and hazard ratio tests. Data mining analysis using CHAID decision tree and logistic regression analysis was applied to define the predictive value of the risk factors of tumor aggressiveness. Our analyses showed, that in invasive LSCC tumors, cells associating with a mesenchymal profile were likely to exhibit enhanced NOS2, TGF-ß, and IL-17A expression levels, concomitantly to NF-κB nuclear translocation. IHC data deciphering determined that EMT induction was also linked to the enrichment of the tumors with CD68+ populations and IL-10 signal. Strikingly, dataset cluster analysis showed that these signatures could define distinct patterns of invasive tumors, where NOS2 associated with IL-10 expression, and TGF-ß and IL-17A signals associated with MMP-9 activation. Decision tree analysis identified IL-17A as a possible predictor of LSCC aggressiveness. Altogether, our results show that distinct immunological patterns would support the acquisition of EMT features in invasive LSCC and suggest that IL-17A may be useful in the early identification of patients "at-risk" of therapeutic failure.

Comparative assessment of HPV, alcohol and tobacco etiological fractions in Algerian patients with laryngeal squamous cell carcinoma.

BACKGROUND: Despite the increasing incidence of laryngeal squamous cell carcinoma (LSCC) in Algeria, scarce information is available on the importance of the preventable etiological factors which may drive the disease. Remarkably, a significant number of cases occur in nonsmoker and nondrinker patients; hence, suggesting that alternative risk factors, like Human papillomavirus (HPV), might be etiologically involved. To gain more insight on the risk factors associated with the disease in the country, we evaluated the etiological fraction of HPV in comparison to tobacco and alcohol intake in LSCC patients. METHODS: To evaluate the etiopathologic fraction (EF) for HPV compared to history of tobacco and alcohol in LSCC, HPV DNA presence in 46 invasive and 3 non-invasive formalin-fixed paraffin-embedded laryngeal tumors was screened using the SPF10-DEIA-LiPA25 Assay. Demographic data and information related to exposure to the risk factors were gathered through interviewer-assisted questionnaires. RESULTS: We observed that 40.8% of all LSCC cases were associated with smoking, 40.8% had combined tobacco and alcohol exposure history, and 14.3% did not show prior exposure to either risk factor. 1 out of 3 in-situ carcinoma cases was positive for HPV-6. HPV prevalence was null in the invasive tumors. HPV DNA was detected in 2.38% for all studied cases. 10.2% of LSCC patients did not associate with any of the studied risk factors. CONCLUSION: Here we show that HPV etiological fraction in LSCC Algerian patients is low and smoking and alcohol remain the principal etiopathologic risk for LSCC burden in Algeria.

IL-6/NOS2 inflammatory signals regulate MMP-9 and MMP-2 activity and disease outcome in nasopharyngeal carcinoma patients.

The role of nitric oxide (NO)(·) in the development of the metastatic properties of nasopharyngeal carcinoma (NPC) is not fully understood. Previous studies proposed that interleukin-6 (IL-6) would act as regulator of matrix metalloprotease activation in NPC. Recently, we showed that (NO)(·) was a critical mediator of tumor growth in patients. The aim of this study was to determine the implication of IL-6 in the progression of NPC pathology via metalloprotease (MMP) activation and their possible correlation with (NO)(·) production. We observed a significant increase in IL-6 and nitrite (NO2 (-)) synthesis in patients (n = 17) as well as a strong expression of IL-6 and nitric oxide synthase 2 (NOS2) in the analyzed tumors (n = 8). In patients' plasma, a negative correlation associated IL-6 with circulating nitrites (r = -0.33). A negative correlation associated the H-scores of these signals in the tumors (r = -0.47). In patients' plasma, nitrite synthesis was positively associated with MMP-9 activation (r = 0.45), pro-MMP-2 expression (r = 0.37), and negatively correlated with MMP-2 activation (r = -0.51). High nitrite levels was associated with better recurrence-free survival (RFS) (p = 0.02). Overall, our results suggest that the IL-6/NOS2 inflammatory signals are involved in the regulation of MMP-9- and MMP-2-dependent metastatic activity and that high circulating nitrite levels in NPC patients may constitute a prognostic predictor for survival.

TNFα antagonization alters NOS2 dependent nasopharyngeal carcinoma tumor growth.

Tumor necrosis factor (TNFα) is a pro-inflammatory cytokine which mediates via nitric oxide (NO) several carcinogenic processes. Increasing evidences suggest that NO promotes inflammation induced growth of nasopharyngeal carcinoma (NPC). In patients, TNFα synthesis associates with poor survival. To explore the effect of the cytokine on NO production and NOS2 dependent NPC growth, NO2(-) (nitrite) producing cells in patients were analyzed in vitro. We observed that patients' monocytes/macrophages (Mo/Ma) and primary tumor biopsies synthesized significant amounts of NO2(-). Interestingly, tumor explants derived NO2(-) levels were more important in elderly patients in comparison with juveniles. Endogenous TNFα neutralization with an anti-TNFα monoclonal antibody (mAb) successfully inhibited NO2(-) synthesis by blood mononuclear cells and tumor explants. Recombinant TNFα (rTNFα) enhanced NO2(-) synthesis and C666-1 NPC cell proliferation. NOS2 selective inhibition (1400W) and TNFα antagonization with an anti-TNFα mAb potently inhibited rTNFα induced C666-1 proliferation and NO2(-) production. Importantly, primary tumors treated with the anti-TNFα mAb also displayed reduced proliferation index (Ki67). Altogether, our results define monocytes/macrophages and the primary tumor as major sources of circulating NO2(-) in NPC patients and support the idea that antibody dependent inhibition of the TNFα/NOS2 pathway may alter NPC tumor growth.

Multicenter evaluation of Neurelec Digisonic® SP cochlear implant reliability.

Over the past decade, the adoption of universal hearing screening in newborns has led to earlier detection of hearing problems and significant lowering of the age of first cochlear implantation. As a consequence, recipients are now expected to keep their cochlear implants (CIs) for a longer period of time. Comprehensive longitudinal information on CI reliability is essential for device choice. The aim of this study was to assess the reliability (in children and adults) of the latest generation of the Digisonic(®) SP CI launched in 2006 by Neurelec. Failure rate (FR) and cumulative survival rate (CSR) for a 5-year period were calculated. This survey is a multicenter retrospective study. A questionnaire was sent to nine CI centers requesting information about patients implanted with Neurelec Digisonic(®) SP CIs. FR and CSR over a 5-year period were calculated on this group. Collaborating centers collected data on 672 patients (362 children and 310 adults) implanted between March 2006 and March 2011. The overall rate of explantation was 2.23 % (15 cases): six devices were explanted due to device failure (0.89 %) and nine were explanted for medical reasons (1.34 %). Four patients were lost to follow-up. The CSR at 5 years was 98.51 % on all patients, 98.48 % for children and 98.57 % for adults. FR was 0.97 % for adults and 0.83 % for children. This first independent study that assesses FR and CSR on the current generation of Digisonic(®) SP CI represents an important resource that can help clinicians and patients during their device choice.

Molecular screening of deafness in Algeria: high genetic heterogeneity involving DFNB1 and the Usher loci, DFNB2/USH1B, DFNB12/USH1D and DFNB23/USH1F.

A systematic approach, involving haplotyping and genotyping, to the molecular diagnosis of non-syndromic deafness within 50 families and 9 sporadic cases from Algeria is described. Mutations at the DFNB1 locus (encompassing the GJB2 and GJB6 genes) are responsible for more than half of autosomal recessive prelingual non-syndromic deafness in various populations. A c.35delG mutation can account for up to 85% of GJB2 mutations and two large deletions del(GJB6-D13S1830) and del(GJB6-D13S1854) have also been reported in several population groups. In view of the genetic heterogeneity a strategy was developed which involved direct analysis of DFNB1. In negative familial cases, haplotype analysis was carried out, where possible, to exclude DFNB1 mutations. Following this, haplotype analysis of five Usher syndrome loci, sometimes involved in autosomal non-syndromic hearing loss, was carried out to identify cases in which Usher gene sequencing was indicated. When homozygosity was observed at a locus in a consanguineous family, the corresponding gene was exhaustively sequenced. Pathogenic DFNB1 genotypes were identified in 40% of the cases. Of the 21 cases identified with 2 pathogenic mutations, c.35delG represented 76% of the mutated alleles. The additional mutations were one nonsense, two missense and one splicing mutation. Four additional patients were identified with a single DFNB1 mutation. None carried the large deletions. Three families with non-syndromic deafness carried novel unclassified variants (UVs) in MYO7A (1 family) and CDH23 (2 families) of unknown pathogenic effect. Additionally, molecular diagnosis was carried out on two Usher type I families and pathogenic mutations in MYO7A and PCDH15 were found.

A new diagnostic marker for secreted Epstein-Barr virus encoded LMP1 and BARF1 oncoproteins in the serum and saliva of patients with nasopharyngeal carcinoma.

PURPOSE: EBV has been associated with nasopharyngeal carcinomas (NPC). In North Africa, the incidence is bimodal-the first peak occurring at approximately 20 years of age and the second peak occurring at approximately 50 years. Standard diagnostic tests based on immunofluorescence using anti-IgA EBV have shown that young North African patients have a negative serology compared with older patients. We are interested in two EBV-encoded oncoproteins, LMP1 and BARF1, which have thus far not been studied in terms of their potential as diagnostic markers for NPC. These two viral oncoproteins have been detected in cell culture media, so we tested whether they could be detected in the serum and saliva of patients with NPC. EXPERIMENTAL DESIGN: LMP1 and BARF1 proteins were analyzed in the sera and saliva of young patients and adult patients with NPC from North Africa and China. We then examined whether the secreted proteins had biological activity by analyzing their mitogenic activity. RESULTS: Both LMP1 and BARF1 were present in the serum and saliva from North African and Chinese patients with NPC. All young North African patients secreted both proteins, whereas 62% and 100% of adult patients secreted LMP1 and BARF1, respectively. From animal studies, the secreted LMP1 was associated with exosome-like vesicles. These secreted EBV oncoproteins showed a powerful mitogenic activity in B cells. CONCLUSION: Both proteins will be a good diagnostic marker for NPC whereas BARF1 is a particularly promising marker for all ages of patients with NPC. Their mitogenic activity suggests their implication in the oncogenic development of NPC.

BAMHI DNA fragment H-polymorphism of Epstein-Barr virus is associated with the mutations present in an 89 BP sequence localized in EBNA2 gene.

To characterize the genotypes of Epstein-Barr virus (EBV) isolate present in North Africa, viruses were isolated from B-lymphoblastoid cell lines established from the saliva of both Algerian Nasopharyngeal Carcinoma (NPC) patients and EBV-positive normal individuals, Algerian Burkitt's lymphoma cell lines, and NPC biopsies. By nucleotide sequence analysis, we showed that there were two specific missense mutations in an 89 bp region of EBNA2 gene at position 49390-49479 of the EBV genome: a mutation at 49449 (C-->A) and another mutation at 49444 (T-->C), changing their amino acid sequence. The first mutation was found in all B cell lines established from the saliva and 50% of BL cell lines, as well as the W91 cell line, while the second mutation was found in EBV isolates from NPC biopsies, BL cell lines and the M-ABA isolate. A PCR-RFLP analysis on the BamHI DNA fragment H showed that the Hl-H2-polymorphism was specifically associated with M-ABA-like mutation, while H-polymorphism was linked with W91-like mutation. The latter was not identified in NPC biopsies, but was found rather in saliva from NPC patients, normal individuals and BL cell lines. The M-ABA-like mutation, on the other hand, was found in 100% of NPC biopsies and some BL cell lines. This suggests that EBV with H1-H2-polymorphism is tightly implicated in NPC development in North Africa rather than EBV with H-polymorphism.